1. Field of the Invention
This invention relates to a measuring method and apparatus using attenuation in total internal reflection such as a surface plasmon sensor for quantitatively analyzing a material in a sample on the basis of generation of surface plasmon, and more particularly to a measuring method and apparatus for measuring on the basis of generation of surface plasmon which is detected as a dark line in measuring light.
2. Description of the Related Art
In metal, free electrons vibrate in a group to generate compression waves called plasma waves. The compression waves generated in a metal surface are quantized into surface plasmon.
There have been proposed various surface plasmon sensors for quantitatively analyzing a material in a sample utilizing a phenomenon that such surface plasmon is excited by light waves. Among those, one employing a system called “Kretschmann configuration” is best known. See, for instance, Japanese Unexamined Patent Publication No. 6(1994)-167443.
The plasmon resonance sensor using the Kretschmann configuration basically comprises a dielectric block shaped, for instance, like a prism, a metal film which is formed on one face of the dielectric block and is brought into contact with a sample, a light source emitting a light beam, an optical system which causes the light beam to enter the dielectric block at various angles of incidence so that total internal reflection conditions are satisfied at the interface of the dielectric block and the metal film and various angles of incidence of the light beam to the interface of the dielectric block and the metal film including an angle of incidence at which attenuation in total internal reflection is generated due to surface plasmon resonance can be obtained, and a photodetector means which detects the intensity of the light beam reflected in total internal reflection at the interface and detects a state of surface plasmon resonance, i.e., a state of attenuation in total internal reflection.
In order to obtain various angles of incidence of the light beam to the interface, a relatively thin incident light beam may be caused to impinge upon the interface while deflecting the incident light beam or a relatively thick incident light beam may be caused to impinge upon the interface in the form of convergent light or divergent light so that components of the incident light beam impinge upon the interface at various angles. In the former case, the light beam which is reflected from the interface at an angle which varies as the incident light beam is deflected may be detected by a photodetector which is moved in synchronization with deflection of the incident light beam or by an area sensor extending in the direction in which reflected light beam is moved as a result of deflection. In the latter case, an area sensor which extends in directions so that all the components of light reflected from the interface at various angles can be detected by the area sensor may be used.
In such a plasmon resonance sensor, when a light beam impinges upon the interface at a particular angle of incidence θsp not smaller than the angle of total internal reflection, evanescent waves having an electric field distribution in the sample in contact with the metal film are generated and surface plasmon is excited in the interface between the metal film and the sample. When the wave vector of the evanescent waves is equal to the wave number of the surface plasmon and wave number matching is established, the evanescent waves and the surface plasmon resonate and light energy is transferred to the surface plasmon, whereby the intensity of light reflected in total internal reflection at the interface of the dielectric block and the metal film sharply drops. The sharp intensity drop is generally detected as a dark line by the photodetector.
The aforesaid resonance occurs only when the incident light beam is p-polarized. Accordingly, it is necessary to set the light beam to impinge upon the interface in the form of p-polarized light.
When the wave number of the surface plasmon can be known from the angle of incidence θsp at which the phenomenon of attenuation in total internal reflection (ATR) takes place, the dielectric constant of the sample can be obtained. That is,
            K      sp        ⁡          (      ω      )        =            ω      c        ⁢                                                      ɛ              m                        ⁡                          (              ω              )                                ⁢                      ɛ            s                                                              ɛ              m                        ⁡                          (              ω              )                                +                      ɛ            s                              wherein Ksp represents the wave number of the surface plasmon, ω represents the angular frequency of the surface plasmon, c represents the speed of light in a vacuum, and ∈m and ∈s respectively represent the dielectric constants of the metal and the sample.
When the dielectric constant ∈s of the sample is known, the concentration of a specific material in the sample can be determined on the basis of a predetermined calibration curve or the like. Accordingly, a property related to the dielectric constant (refractive index) of the sample can be detected by detecting the angle of incidence θsp at which the intensity of light reflected in total internal reflection from the interface of the prism and the metal film sharply drops (this angel θsp will be referred to as “the attenuation angle θsp”, hereinbelow).
In such a surface plasmon sensor, it has been proposed, in order to measure the attenuation angle θsp accurately with a wide dynamic range, to use a photodetector in the form of an array of a plurality of photodetector elements arranged in a predetermined direction so that light beam components reflected at different angles at the interface impinge upon different photodetector elements as disclosed in Japanese Unexamined Patent Publication No. 11(1999)-326194.
In this case, the output signals output from the photodetector elements are generally differentiated in the direction in which the photodetector elements are arranged, and the refractive-index-related property of the material to be measured is generally obtained on the basis of the differentials.
As a similar apparatus utilizing the phenomenon of attenuation in total internal reflection (ATR), there has been known a leaky mode sensor described in, for instance, “Spectral Research” Vol.47, No.1 (1998), pp21 to 23 & pp26 and 27. The leaky mode sensor basically comprises a dielectric block shaped, for instance, like a prism, a clad layer which is formed on one face of the dielectric block, an optical waveguide layer which is formed on the clad layer and is brought into contact with a sample, a light source emitting a light beam, an optical system which causes the light beam to enter the dielectric block at various angles of incidence so that total internal reflection conditions are satisfied at the interface of the dielectric block and the clad layer and various angles of incidence of the light beam to the interface of the dielectric block and the clad layer including an angle of incidence at which attenuation in total internal reflection is generated due to optical waveguide mode excitation can be obtained, and a photodetector means which detects the intensity of the light beam reflected in total internal reflection at the interface and detects a state of waveguide mode excitation, i.e., a state of attenuation in total internal reflection (ATR).
In the leaky mode sensor with this arrangement, when the light beam is caused to impinge upon the clad layer through the dielectric block at an angle not smaller than an angle of total internal reflection, only light having a particular wave number and impinging upon the optical waveguide layer at a particular angle of incidence comes to propagate through the optical waveguide layer in a waveguide mode after passing through the clad layer. When the waveguide mode is thus excited, almost all the incident light is taken in the optical waveguide layer and accordingly, the intensity of light reflected in total internal reflection at the interface of the dielectric block and the clad layer sharply drops. That is, attenuation in total internal reflection occurs. Since the wave number of light to be propagated through the optical waveguide layer in a waveguide mode depends upon the refractive index of the sample on the optical waveguide layer, the refractive index and/or the properties of the sample related to the refractive index can be detected on the basis of the angle of incidence at which the attenuation in total internal reflection occurs.
Also in such a leaky mode sensor, a photodetector in the form of an array of a plurality of photodetector elements can be used to detect the position of the dark line generated due to the attenuation in total internal reflection, and at the same time differentiation of the output signals output from the photodetector elements is often applied.
The surface plasmon sensor and the leaky mode sensor are sometimes used in random screening for finding a specific material combined with a predetermined sensing material in the field of pharmacy. In this case, a sensing material is fixed on the film layer (the metal film in the case of the surface plasmon sensor and the clad layer and the optical waveguide layer in the case of the leaky mode sensor), and a sample liquid containing a material to be analyzed is spotted on the sensing material. Then the attenuation angle θsp is repeatedly measured each time a predetermined time lapses.
When the sample material (the material to be analyzed in the sample liquid) is combined with the sensing material, the refractive index of the sensing material changes with time due to combination with the sample material. Accordingly, by measuring the attenuation angle θsp, at which attenuation in total internal reflection takes place, for every predetermined time, thereby detecting whether the attenuation angle θsp changes (to know the state of combination of the sample material with the sensing material), it is possible to know whether the sample material is a specific material to be combined with the sensing material. As combinations of such a specific material and a sensing material, there have been known combinations of an antigen and an antibody and of an antibody and another antibody. For example, rabbit antihuman IgG antibody is fixed on the surface of the film layer as the sensing material with human IgG antibody employed as the specific material.
In order to detect the state of combination of the sample material with the sensing material, the total reflection attenuation angle θsp (the angle of incidence θsp at which attenuation in total internal reflection takes place) itself need not necessarily be detected. For example, the amount of change in the total reflection attenuation angle θsp after the sample liquid is spotted onto the sensing material is measured and the state of combination of the sample material with the sensing material may be measured on the basis of the amount of change of the total reflection attenuation angle θsp. When a photodetector in the form of an array of a plurality of photodetector elements and differentiation of the output signals output from the photodetector elements are employed, the state of combination of the sample material with the sensing material can be measured on the basis of the amount of change of the differentiation of the output signals. (See our Japanese Patent Application No. 2000-398309.)
In the practical apparatuses utilizing the phenomenon of attenuation in total internal reflection such as a surface plasmon sensor or a leaky mode sensor, the amount of change of the total reflection attenuation angle θsp is measured by spotting a sample liquid comprising solvent and a sample material onto a film layer formed on the bottom of a measuring chip in the form of a cup or dish.
This applicant has disclosed a measuring apparatus utilizing the phenomenon of attenuation in total internal reflection in which a plurality of measuring chips are placed on, for instance, a turn table and are measured in sequence, thereby shortening the time required to measure a lot of samples. (Japanese Unexamined Patent Publication No. 2001-330560)
Further, this applicant has disclosed a measuring apparatus utilizing the phenomenon of attenuation in total internal reflection in which measuring chips each having a plurality of sample liquid holding portion are used so that measurement on a plurality of samples can be done at one time without moving the measuring chips. (Japanese Patent Application No. 2001-397411)
In measuring apparatuses utilizing the phenomenon of attenuation in total internal reflection, it is necessary to keep constant the environmental temperature throughout measurement since the refractive index of the sample is changed depending on the temperature around the dielectric block.
However, since the dielectric block is kept exposed to a light beam, the temperature around the dielectric block is raised, which changes the refractive index of the sample and causes drift of the detecting signal.
Further, when the film layer (the metal film in the case of the surface plasmon sensor and the clad layer and the optical waveguide layer in the case of the leaky mode sensor) is non-uniform in thickness, or the sensing material fixed on the film layer is non-uniform in reactivity, or when dust adhere to the film layer, the result of measurement can fluctuate according to the position where the light beam impinges upon the interface. The sensing material described above is discussed in detail, for instance, in our Japanese Patent Application 2001-113647.
The problem of fluctuation in the result of measurement may be overcome by causing the light beam to impinge upon the interface between the film layer and the dielectric block not to be focused thereon as disclosed in Japanese Patent Application 2000-149415. That is, when the light beam impinges upon the interface over an area of a certain width, measurement can be done under a condition where the thickness of the film layer, the reactivity of the sensing material and the like are averaged and fluctuation in the result of measurement can be suppressed.
However, when the light beam is not focused on the interface and impinges upon the interface over an area of a certain width, the range of angle of incidence of the light beam at which the attenuation in total internal reflection takes place is widened and the measuring sensitivity deteriorates.
Further, the problem of fluctuation in the result of measurement may be overcome by preparing a plurality of measuring units, each comprising a dielectric block and a film layer, for one sample, and carrying out measurement on one sample a plurality of times using different measuring units, and statistically processing the results of measurement. However, this approach is disadvantageous in that it takes a long time to obtain result of measurement for one sample.
Further in the case where a photodetector in the form of an array of a plurality of photodetector elements arranged in a predetermined direction is employed to detect the position of a dark line as disclosed in Japanese Unexamined Patent Publication No. 11(1999)-326194, the output characteristics versus the position of the dark line becomes non-linear and it becomes difficult to perform exact measurement when the photodetector elements of the photodetector are not uniform in sensitivity or the sensitivity characteristics of the photodetector elements are non-linear.
Conventionally, the intensity of the light beam reflected at the interface is generally detected by a photodetector which is moved in synchronization with deflection of the light beam or an area sensor such as a CCD having a large light receiving face. However, the former is disadvantageous in that since it requires a mechanical drive mechanism, it is not suitable for analyzing samples at high speed though a wide dynamic range can be ensured with respect to the measuring range of the attenuation angle θsp, whereas the latter is disadvantageous in that since the area sensor is low in resolution and dynamic range for charge accumulation, it is difficult to ensure high analyzing accuracy though being suitable for analyzing samples at high speed.
In order to avoid the difficulties described above, we, this applicant, have proposed a measuring apparatus in which the intensity of the light beam reflected at the interface is detected by a photodetector means comprising a plurality of photodetector elements, the outputs of adjacent pairs of photodetector elements of the photodetector means are differentiated and the attenuation angle θsp is obtained on the basis of the differentials as disclosed, for instance, in Japanese Unexamined Patent Publication No. 11(1999)-326194. This apparatus can detect the attenuation angle θsp more accurately with a wider dynamic range as compared with area sensors like a CCD.
Recently, there has been a demand in apparatuses such as that disclosed in Japanese Unexamined Patent Publication No. 11(1999)-326194 that the profile of the light beam detected by the photodetector means is to be known. However, in such a measuring apparatus, each of the photodetector elements is large in size in order to increase the output signal of the photodetector element. That is, the photodetector elements are arranged at rough pitches, which results in low resolution of the beam profile.
Whereas when the photodetector elements are arranged at fine pitches in order to detect the beam profile in a high resolution, the output signal of each photodetector element becomes too weak to accurately obtain the attenuation angle θsp. Further, when the amount of light received by each photodetector element is small, influence of noise is strengthened.